Spermatogonial stem cells (SSCs) are unique in that they are adult cells poised on the threshold of pluri- potency, having the ability to become spontaneously reprogrammed to pluripotency in vitro without addition of exogenous factors. SSC culturing and the reprogramming of SSCs into pluripotent cells are two new technologies that hold great promise to create patient-specific therapies for treating myriad diseases. Our long-term goals are to elucidate the molecular mechanisms underlying SSC self-renewal and differentiation and the phenomenon of SSC reprogramming, and to translate these discoveries into cell-based therapeutics. However, the rarity of the true stem cells in SSC cultures and the inefficiency of SSC reprogramming are major impediments to attaining the therapeutic goals. Here we test a strategy for overcoming these impediments through manipulation of the transcription factor, OCT4, while examining fundamental questions of SSC regulation. Exquisite control of OCT4 levels is critical for maintaining pluripotency in embryonic cells with aberrations leading to differentiation. Based on our studies on OCT4 function in mouse SSCs and an emerging model for post-translational control of OCT4 in ES cells we propose the following hypothesis: SSC fate is determined in part by fine-tuning OCT4 protein levels through post-translational modification. The first aim is to determine how OCT4 levels affect SSC fate in vitro and is directly relevant to achieving SSC-based therapy. Preliminary data using SSCs engineered to overexpress OCT4 in a Doxycycline dose-dependent manner suggest that in- creased OCT4 is sufficient to increase reprogramming; also, increased OCT4 suppresses SSC differentiation in vitro and the prediction is that SSC numbers are correspondingly increased. The second aim is to examine mechanisms for regulating OCT4 protein levels in SSCs. We will identify cis- and trans-acting factors controlling OCT4 ubiquitination beginning by testing the function of the E3 ubiquitin ligase, Wwp2, in SSCs. The third aim is to determine how aberrant OCT4 levels affect SSC fate in vivo. By creating a transgenic mouse to conditionally overexpress OCT4, we will test the idea that OCT4 levels are turned down during spermatogenesis to permit spermatogonial differentiation, a process that remains ill-defined. Also, OCT4 overexpression is linked to oncogenesis and our mouse model will be useful to determine whether OCT4 overexpression plays a causitive role in germ cell tumorogenesis. This proposal's highly innovative aspects are: (1) improving reprogramming efficiency with stabilized OCT4; (2) a focus on SSC reprogramming rather than somatic cell reprogramming; (3) analysis of OCT4 function and regulation in SSCs rather than ES cells. Our studies will impact fundamental understanding of the relationship between SSCs, pluripotent cells and the tumorigenic cells that may result from misregulation of spermatogonial fate. Furthermore, the studies are significant because they will provide a foundation of knowledge and mechanistic insight that will be applicable to increasing the percentage of stem cells in SSC cultures and the frequency of reprogramming through regulation of OCT4 protein. PUBLIC HEALTH RELEVANCE: A strategy for regenerative medicine is envisioned wherein spermatogonial stem cells (SSCs) could be isolated from the testes and used in patient-specific therapy either directly, to replenish testicular germ cells following chemotherapy, or indirectly, y reprogramming SSCs to other types of cells prior to transplantation for treatment of a variety of diseases. Understanding the molecular mechanisms governing SSC fate and the reprogramming process is a crucial step to achieving this vision and will also be relevant to treating infertility and testicular cancer.